/. Biochem. 86, 991-1000 (1979)

A Novel Peptidoglycan-Associated Lipoprotein Found in the Cell Envelope of Pseudomonas aeruginosa and Escherichia coli

Takeshi MIZUNO Mitsubishi-Kasei Institute of Life Sciences, Machida, Tokyo 194 Received for publication, April 2, 1979

Protein H, one of the major outer membrane proteins of Pseudomonas aeruginosa, shows an interesting interaction with the peptidoglycan layer of the cell. It is retained by peptidoglycan after extraction of the cell envelope with SDS solution at 35°C. A protein of the same molecular weight (21,000) as protein H was found in the peptidoglycan-associated fraction of Escherichia coli K-12 prepared under the same conditions. This protein, designated here as protein 21K, was purified from the cell envelope of E. coli, and the properties of two proteins (protein H and protein 21K) were compared. By gas chromatographic analysis of purified protein 21K and protein H, it was found that both contained covalently linked fatty acid. In isotopic labeling experiments, [14C]palmitic acid and [2-3H]glycerol were incorporated into both proteins H and 21K. These two proteins showed similar amino acid compositions, but no apparent correlation was found between protein 21K or protein H and Braun's lipoprotein. These results suggest that protein 21K off. coli K-12 and protein H of P. aeruginosa PAO are a novel type of lipoprotein. All nine gram-negative bacteria tested, including enteric and non-enteric bacteria, contained a similar protein of apparent molecular weight 21,000 as a peptidoglycan-protein complex.

The outer membrane of gram-negative bacteria consists of proteins, phospholipids, and lipopolysaccharide. E. coli outer membrane proteins have been characterized by several authors, and some of them were found to have a particular interaction with peptidoglycan. They are the so-called peptidoglycan-associated proteins (J-3), and Braun's lipoprotein (4). Recently, Hazumi et al. reported two new peptidoglycan-associated proteins (0-13, 0-14) in the outer membrane of E. coli K-12 (5). We have reported that the outer membrane of P. aeruginosa contains at least six major proteins (proteins D-I) (6). Recently we

found protein I was analogous to Braun's lipoprotein of E. coli (7), and that two major outer membrane proteins (protein F and protein H) were associated with peptidoglycan (8). Braun's lipoprotein of E. coli contains a covalently-linked lipid at the amino-terminal of the polypeptide (4). As for as we know, this is the only lipoprotein so far described in the outer membrane of gram-negative bacteria. In this paper, we report evidence for the presence of a novel peptidoglycan-associated lipoprotein in the cell envelopes of E. coli and P. aeruginosa.

Abbreviation: SDS, sodium dodecyl sulfate. Vol. 86, No. 4, 1979

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T. MIZUNO

SDS buffer overnight, and applied to an SDSSephacryl S-200 superfine column (2.6 x 85 cm) MATERIALS AND METHODS previously equilibrated with SDS buffer. The gel Strains and Growth Conditions—Escherichia filtration was carried out at a flow rate of 35 ml/h, coli K-12 W3110 and Pseudomonas aeruginosa and 3.5-ml fractions were collected. Peak fracPAO1 and PAO3012 (Trp") were used in most tions of protein 21K or Braun's lipoprotein were of the present studies. The following gram- treated with cold acetone as described above. negative bacteria were also used. Escherichia coli The preparation enriched in protein 21K was BB was obtained from Dr. T. Shiba; Salmonella dissolved in Triton X-100-urea buffer [2% Triton typhimurium \TTL from Dr. T. Nakae; Klebsiella X-100-6M urea-lOmM Tris-HCl (pH 7.4)], then aerogenes from Dr. Y. Murooka; Serratia mar- dialyzed against the same buffer at 20°C overnight. cescens Sb from Dr. T. Tanaka; Pseudomonas The sample was chromatographed on a DEAEputida Cl S from Dr. K. Nagahari; Proteus vulgaris cellulose column (1x40 cm) previously equiliIAM2003 from the Institute of Applied Micro- brated with Triton X-100-urea buffer at a flow biology (the University of Tokyo); Pseudomonas rate of 12.3 ml/h. The column was washed with fluorescens ATCC17397 from our laboratory 90 ml of the same buffer and eluted with 200 ml of collection. a linear gradient of 0-0.2 M NaCl in the same buffer. Nutrient broth and glutamate medium (9) If necessary, the same DEAE-cellulose column chromatography was repeated. The fractions were used in most experiments. Preparation of Cell Envelope and Outer Mem- containing pure protein 21K were treated with brane—Cell envelopes and outer membranes were cold acetone as described above and the precipitates were dispersed in distilled water. The preparation prepared as described previously (7). was dialyzed against distilled water and stored at SDS Extraction of Cell Envelope—SDS differ-20°C or -70°C. ential extraction of cell envelopes was carried out as described previously, unless otherwise noted (8). The preparation enriched in Braun's lipoIsolation of Protein H from P. aeruginosa and protein was further purified by re-chromatography Protein 2IK from E. coli—The procedures for the with Sephacryl S-200 under the conditions described purification of protein F and protein H from P. above. aeruginosa PAO3012 were described in detail in Analytical Methods—Amino acid analysis was the preceding paper (8). Procedures for the performed as described previously (7). Fatty acid purification of protein 21K from E. coli K-12 content was determined with a Shimadzu GCW3110 were as follows. Cell envelopes prepared 4BM gas chromatograph using 2-5 mg of protein from E. coli (50 g wet weight) grown in nutrient with pentadecanoic acid methyl ester as an internal broth were treated with 360 ml of 2% SDS-10% standard as described previously (7). Protein glycerol-0.15 M NaCl-10 mM Tris-HCl (pH 7.8) at concentrations were determined by amino acid 30°C for 60 min. The insoluble fraction was analysis of the hydrolysates, assuming the molecollected by centrifugation at 100,000 xg for 60 cular weights of protein 21K and protein H to be min. The insoluble fraction was washed once 21,000(7,5). with 220 ml of 2% SDS-10% glycerol-10 mM SDS-Polyacrylamide Gel Electrophoresis— Tris-HCl (pH 7.8) at room temperature. The Urea-SDS, and SDS-polyacrylamide slab gel peptidoglycan-protein complex thus obtained was electrophoreses were carried out as described re-extracted with 200 ml of 2% SDS-10% glycerol- previously (6). 10 mM Tris-HCl (pH 7.8) at 50°C for 60 min. Radioisotope Labeling of Peptidoglycan-AsProteins were precipitated from the supernatant by sociated Proteins—E. coli K-12 (W3110) was labeled the addition of two volumes of cold acetone in with 15 pC\ of [l-14C]palmitic acid (55.3 mCi/mmol) the presence of 10 mM MgCl2. The precipitate in 30 ml of nutrient broth, or with 75 fid of [4,5was washed once with 90% cold acetone and 3H]leucine (21.9 mCi/mmol) in 30 ml of glutamate dissolved in SDS buffer [1 % SDS-5 mM EDTA- medium for three generations. Cell envelopes 50 mM NaCl-0.02% NaN3-25 mM sodium phos- were prepared from both labeled cells and comphate (pH 7.2)]. The sample was dialyzed against bined. P. aeruginosa PAO1 was also labeled /. Biochem.

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993

CELL ENVELOPE OF P. aeruginosa and E. coli under the same conditions. The cell envelopes from E. coli were treated with 2 % SDS-lOmM Tris-HCl (pH7.8)-10% glycerol-0.1 M NaCl for 40 min at 30°C. The insoluble fraction was extracted with the same solution without 0.1 M NaCl for 60 min at 50°C. The cell envelopes from P. aeruginosa were treated with 2 % SDS-10 mM Tris-HCl (pH7.8)-10% glycerol for 40 min at 30°C. The insoluble fraction was extracted with the same solution for 60 min at 80°C. To the extracts (500 ft\), 1 ml of acetone and 200 [i% of bovine serum albumin were added, and the mixture was kept overnight at 4°C. Phospholipids were extracted extensively from the precipitates with chloroform : methanol ( 2 : 1 ) four times. Analyses of labeled proteins by urea-SDS polyacrylamide gel electrophoresis were carried out as described previously (7). For [2-3H]glycerol labeling, E. coli or P. aeruginosa was labeled with 200 /*Ci of [2-3H]glycerol (29.1 mCi/mmol) in 30 ml of nutrient broth for two generations, or with 20 ^Ci of [14C]leucine (5.8 mCi/mmol) in 30 ml of glutamate medium for three generations. Other procedures were the same as those for [l4C]palmitic acid labeling. RESULTS Isolation of Protein H from P. aeruginosa and Protein 21K from E. coli—When the cell envelope of P. aeruginosa was extracted with 2 % SDS solution at 35°C, two proteins (protein F and protein H) were retained by peptidoglycan (Fig. 1A, gel c) as described in the preceding paper (§). Under the same extraction conditions, E. coli cell envelopes retained several proteins including matrix proteins (0-8/0-9) and Braun's lipoprotein as had been reported by several investigators (1-3, JO) (Fig. IB, gel h). Figure IB shows that the peptidoglycan-protein complex of E. coli contained another protein with the same apparent molecular weight (21,000) as protein H of P. aeruginosa. We tentatively designated this protein as protein 21K. Purification of protein H from P. aeruginosa was described in the preceding paper (8). Purification of protein 21K from E. coli was carried out by differential extraction of cell envelopes with SDS solution at two different temperatures (first, 30°C; second, 50°C) as described in " MATERIALS AND METHODS." Protein 21K and the free

P.aeruginosa a b c d e

-0-8/0-9

H-

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—21K

1

Braun's Lipoprotein

Fig. 1. SDS-polyacrylamide gel electrophoresis of peptidoglycan-associated proteins from P. aeruginosa PAO3012 and E. coli K-12 (W3110). A, P. aeruginosa PAO3012; B, E. coli K-12 (W3110). (a) and (f), cell envelopes; (b) and (g), 2% SDS-soluble fractions of cell envelopes; (c) and (h), 2% SDS-insoluble fractions of cell envelopes; (d), purified protein F; (e), purified protein H; (i), purified protein 21K; (j), purified Braun's lipoprotein. All samples were boiled for 5 min except in the case of (d), which was treated at 30°C for 30 min. form of Braun's lipoprotein were recovered in the soluble fraction at the second extraction (at 50°C), whereas 0-8/0-9 were retained in the insoluble fraction even after these two steps of extraction. Protein 21K and Braun's lipoprotein were separated from each other by SDS-Sephacryl S-200 column chromatography (Fig. 2). Further purification of protein 21K was carried out by Triton X-100urea DEAE-cellulose ion-exchange chromatography as shown in Fig. 3. The purified preparations of protein H from P. aeruginosa PAO and protein 2IK. from E. coli K-12 showed the same mobility on SDS-polyacrylamide gel electropherograms (Fig. 1). Protein H and Protein 2IK Contain Fatty Acids—We found that purified proteins H and 21K contain covalently linked fatty acids. Braun's lipoprotein of E. coli and protein F of-P. aeruginosa were also examined for comparison (Fig. 1). Purified protein H or 21K was extracted extensively with chloroform : methanol (2 : 1) to eliminate possible phospholipid contamination. These dephospholipidated proteins were analyzed by gas chromatography. As shown in Fig. 4, fatty acids were found in protein H and protein 21K. As a positive control, purified Braun's lipoprotein was

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T. MIZUNO 1

0.5

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b

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200 250 300 350 Elution Volume ( m l )

Fig. 2. Isolation of protein 21K from E. coli K-12 (W3110) by SDS-Sephacryl S-200 superfine column chromatography. The procedures are described in " MATERIALS AND METHODS." The inset electropherograms show the protein compositions of (a), peak 21K and (b), peak BLP. Abbreviation: BLP, Braun's lipoprotein.

acid methyl ester as an internal standard (Fig. A). In this experiment, Braun's lipoprotein was found to contain 2.6 mol of fatty acids per mol of protein, which is consistent with Braun's result (4). Among the fatty acids found in protein 21K and protein H, hexadecanoic acid (Ci6:0) was predominant, accounting for 80% to 90% of total fatty acids. [liC]Palmitic Acid Incorporation—To confirm that protein 21K and protein H contain fatty acids, 100 200 300 labeling experiments with [14C]palmitic acid were Elution Volume(ml) carried out. Peptidoglycan-protein complexes Fig. 3. Elution profile of protein 21K from E. coli were isolated from the cell envelope of E. coli 14 (W3110) on Triton X-100-urea DEAE-cellulose column K-12 and P. aeruginosa PAO1 labeled with [ C]3 chromatography. The procedures are described in palmitic acid and [ H]leucine. As shown in Fig. " MATERIALS AND METHODS." The inset electro- 5, the [3H]leucine radioactivity and the [14C]pherogram shows the protein composition of the shaded palmitic acid radioactivity were superimposed in the peaks of both protein 21K and protein H. These radioactivities were also superimposed in the also analyzed (Fig. 4), since it has been established free form of Braun's lipoprotein14of E. coli (Fig. 5 A). that Braun's lipoprotein contains three fatty acid These results indicate that [ C]palmitic acid is residues per mol of protein (4, 11). As a negative specifically incorporated into protein 21K and control, purified protein F of P. aeruginosa was protein H as well as E. coli. Braun's lipoprotein, proteins contain fatty acids. also analyzed. No significant amount of fatty and suggest that these 14 The other large [ C]peaks found in Fig. 3B are acids was found in protein F (data not shown). probably due to lipopolysaccharide. The amounts of total fatty acids per mol of protein were calculated to be 2.7 and 2.6 for protein 21K [2-3H]Glycerol Incorporation—We investiand protein H, respectively, with pentadecanoic gated whether protein 21K. and protein H contain ./. Biochem.

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CELL ENVELOPE OF P. aeruginosa and E. coli A.

Protein H

B.

( P aeruginosa)

995 Protein 21 K

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Retention Time(min) Fig. 4. Gas chromatograms of fatty acids isolated from peptidoglycan-associated proteins of P. aeruginosa PAO3012 and E. coli K.-12 (W3110). After hydrolysis of (A), protein H; (B), protein 21K; and (C), Braun's lipoprotein, fatty acids were isolated and converted to their methyl esters. Pentadecanoic acid methyl ester (C15;o) was added as an internal standard (IS) prior to hydrolysis. For the identification of fatty acids, authentic fatty acid methyl esters (C14:0, C, 6:0 , C le:1 , C I8:0 , Ci8:1) from Sigma were used as standards. Details are given in (7).

any glycerol residue. Peptidoglycan-protein complexes were isolated from the cell envelope of E. coli K-12 and P. aeruginosa PAO1 labeled with [2-3H]glycerol and [14C]leucine. As shown in Fig. 6, [2-3H]glycerol was incorporated into protein 21K and protein H as well as Braun's lipoprotein of E. coli. Taking the relative ratio of [3H] and [14C]radioactivity of Braun's lipoprotein as 1.00, that of protein 21K was calculated to be 0.34 (Fig. 6A). On the other hand, the molar ratio of leucine of Braun's lipoprotein to protein 21K was calculated to be 0.33 based on amino acid analysis (Table I). Based on these results, it is estimated that protein 21K contains one glycerol residue per mol of protein, since the glycerol content of Braun's lipoprotein is known to be one

mol of glycerol per mol of protein (4, 11). The glycerol content in protein H of P. aeruginosa could not be estimated in the same way, since protein I, which is analogous to Braun's lipoprotein of E. coli (7), was not retained in the peptidoglycan-protein complex (8). These results suggest that protein 21K and protein H contain a glycerol residue. Amino Acid Analysis of Protein 21K and Protein H—The amino acid compositions of protein 21K from E. coli and protein H from P. aeruginosa are listed in Table I. The distributions of amino acids of protein 21K and protein H were broadly similar. Both proteins contained no detectable cysteine. For comparison, the results of amino acid analysis of Braun's lipoprotein from E. coli

Vol. 86, No. 4, 1979

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T. MIZUNO

996

A. E.coli

0

20 Slice

40 60 Number

80

20 40 60 Slice Number

80

Fig. 5. Urea-SDS polyacrylamide gel electrophoresis of the peptidoglycan-associated proteins labeled with [3H]leucine and [14C]palmitic acid. A, E. coli K-l2 (W3110); B, P. aeruginosa PAO1. , [3H]Leucine, , [14C]palmitic acid. Gels were sliced into 1 mm widths and counted (7). Details are given in " MATERIALS AND METHODS." Arrows with letters indicate the positions of the individual proteins determined by parallel runs of purified outer membranes, protein F, protein H, protein 21K, and Braun's lipoprotein. It should be noted that heat-modifiable protein F of P. aeruginosa was converted to protein F* under these conditions (6). Abbreviations: BLP, Braun's lipoprotein, BPB, bromphenol blue.

and protein I from P. aeruginosa are also listed in Table I. The present amino acid composition of Braun's lipoprotein of E. coli coincides well with that established by Braun and Bosch, except for glycine {12, 13). No apparent correlation was found between the amino acid compositions of protein 21K and Braun's lipoprotein of E. coli or between those of protein H and protein I of P. aeruginosa (Table I). These results suggest that protein 21K of E. coli and protein H of P. aeruginosa are a novel type of lipoprotein, different from Braun's lipoprotein. Occurrence of Peptidoglycan-Associated Protein 21K in Gram-Negative Bacteria—To examine whether a similar peptidoglycan-associated protein exists in other gram-negative bacteria, peptidoglycan-protein complexes were prepared by SDS

extraction at 35°C from cell envelopes of nine gram-negative bacteria. All six Enterobacteriaceae tested (Escherichia coli K-l2, Escherichia coli BB, Salmonella typhimurium LT2, Klebsiella aerogenes W70, Serratia marcescens Sb, and Proteus vulgaris IAM2003) and three other gram-negative bacteria (Pseudomonas aeruginosa PAO, Pseudomonas fluorescens ATCC17397, and Pseudomonas putida CIS) contained a protein with an apparent molecular weight of 21,000 in their peptidoglycanprotein complexes (Fig. 7A). Proteus seems to be extremely rich in this protein, though its apparent molecular weight was a little smaller (18,000). Under the present extraction conditions, all nine bacteria tested contained one or more peptidoglycan-associated proteins with apparent molecular weights between 33,000 and 40,000, which probably correspond to Rosenbusch's

J. Biochem.

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997

CELL ENVELOPE OF P. aeruginosa and E. coli

40 60 Number Fig. 6. Urea-SDS polyacrylamide gel electrophoresis of the peptidoglycan-associated proteins labeled with [14C]leucine and [2-3H]glycerol. A, E. coli K.-I2 (W3110); B, P. aeruginosa PAO1. , [14C]Leucine; 3 , [2- H]glycerol. Other details were the same as in the legend to Fig. 5. TABLE I. protein I.

Comparison of the amino acid compositions of protein 21K, protein H, Braun's lipoprotein and

Residues per polypeptide mol/mola Mminu . acid Asp Thr« Sere Glu Pro Gly Ala 1/2-Cysf Val Met' lie Leu Tyr Phe His Lys Arg TrpK a Hydrolysis assumed for

A novel peptidoglycan-associated lipoprotein found in the cell envelope of Pseudomonas aeruginosa and Escherichia coli.

/. Biochem. 86, 991-1000 (1979) A Novel Peptidoglycan-Associated Lipoprotein Found in the Cell Envelope of Pseudomonas aeruginosa and Escherichia col...
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